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Repeated Cross-fostering Protocol as a Mouse Model of Early Environmental Instability
重复交叉抚育实验建立早期环境不稳定的小鼠模型   

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Abstract

Early life events have a crucial role in programming the individual phenotype indeed the exposure to traumatic experiences during infancy can increase later risks for a variety of neuropsychiatric conditions, including mood and anxiety disorders. Several studies in rodents demonstrated the impact of short and long sessions of separation/isolation from caregivers in developing pups, on the behavioral and hormonal response to stress during infancy and adulthood (D’Amato et al., 1998; Meaney et al., 2000; Luchetti et al., 2015). The repeated cross-fostering (RCF) is an early manipulation carried out in mouse pups during the first four postnatal days life. Differently from other early manipulations, hypotalamic-pituitary-adrenal (HPA) axis functioning is not altered in RCF treated subjects. This manipulation is used to model human early environmental instability, a risk factor for internalizing disorders including separation anxiety disorder, panic disorder and CO2 hypersensitivity (Kendler et al., 1992; Forman and Davies, 2003; Battaglia et al., 2009).

Keywords: Cross-fostering(交叉培养), Early environment(早期的环境), Mother-pups bond(母亲只债券)

Materials and Reagents

Note: See Figure 1.


Figure 1. Materials and equipment. a. paper towels for cage enrichment; b. clean cage; c. hot plate; d. balance; e. latex gloves; f. tags for mum and pups identification.

  1. Paper towels for cage enrichment 

  2. Gloves to manipulate animals (latex)

  3. Balance
  4. 10 females (minimum number) and 5 males of the same mouse strain (we use NMRI, C57BL6 and DBA, but it is possible to use this protocol in other strains) for mating 


Equipment

Note: See Figure 1.

  1. Clean cages to isolate pregnant females and to temporarily maintain pups on the hot plate during the manipulation, as described at point 2 of "procedure step by step" (washed transparent high temperature polysufone cages 26.7 x 20.7 x 14.0 cm, with clean bedding)

Procedure


  1. Mating procedure consists of in housing 2 females with one male for 15 d. The experimental procedure requires that, at least 5 females give birth simultaneously, within 24 h. To reach this goal and synchronize females’ estrous cycles, females can be exposed to male odor a few days before mating. Check female weight at mating.

  2. On the 15th day, males are removed and females are weighed to assess pregnant status. The weight gained, suggesting pregnancy, depends on the strain of mice used, but generally is between 10 and 15 grams in 15 days (a 50% increase of weight from mating day); in addition visible lateral "bulges" are visible in pregnant females.
  3. Each pregnant female is isolated in a clean cage with bedding material (paper) and daily checked for pups’ birth (PND0: postnatal day 0).

  4. At least 5 females should give birth within 24 h to permit the execution of RCF manipulation.

Rcf manipulation
Pups spend the first postnatal day (PND0) with the biological mother. Differently from the ‘‘classical’’ cross-fostering procedures (Bartolomucci et al., 2004), RCF pups change caregiver every 24 h for 4 times during the PND1-PND4 time interval, by following a rotation scheme: Each dam receives 4 different litters during this manipulation and each litter changes 4 different adoptive dams (the adoptive dam is the mum who receive pups from another mum) (also see Figure 2 and the Table 1). The daily procedure consists of first removing the mother from the cage, then removing its entire litter, and immediately introducing this litter into the home-cage of a different dam whose pups had just been removed. The RCF pups were then semi-covered with the home-cage bedding of the adoptive mother, which is then reintroduced in the cage and left with this litter for the next 24 h. The entire procedure lasted about 30 sec and took place every day at same time during the light cycle of a standard light/dark cycle (usually between 10.30 and 11.00 a.m.). This was repeated daily, four times until reaching the fourth adoptive mother, with which pups were left until weaning (PND 28).
Adoptive dams should be lactating females with pups of the same age as fostered litters and litters should have same or at least similar number of pups to avoid possible nutritional stress for mothers and pups.

The control manipulation consists in first removing the mother from the cage, then picking up control pups, reintroducing them in their home-cage and covering with home-cage bedding. At the end of this manipulation, which lasts 30 sec, the biological mother is reintroduced in the home cage. This control procedure takes place daily from PND1 to PND4, to assess possible effects of the handling procedure necessarily required by RCF protocol. It is not necessary that control females give birth exactly on the same day.

Procedure step by step
For example, we consider 5 litters RCF and one CONTROL. We identify RCF mums with uppercase letters A, B, C, D and E, and respective pups with the same but lowercase letters a, b. c, d, and e. The control mum identification is F and respective pups f.
To facilitate the procedure we record mum’s code on the home cage tag while the pups’ code is written on a smaller removable tag that can be transferred (together with pups) to the adoptive mother’s home-cage tag.
First day (PND1)

  1. Remove mum “A” from its home cage.
  2. Remove pups “a” from the home cage and put them in a clean cage on hot plate (30-35 °C) to maintain body 
temperature.
  3. Remove mum “B” from its home cage. 

  4. Move pups “b” from their home cage to the mum’s “A” cage, semi-cover pups with bedding and reintroduce 
mum “A” in its home cage. 

  5. Remove mum “C” from its home cage.
  6. Move pups “c” from their home cage to the mum’s “B” cage, cover pups with some bedding and reintroduce 
mum “B” in its home cage.
  7. Remove mum “D” from its home cage. 

  8. Move pups “d” from their home cage to the mum’s “C” cage, cover pups with some bedding and reintroduce 
mum “C” in its home cage. 

  9. Remove mum “E” from its home cage. 

  10. Move pups “e” from their home cage to the mum’s “D” cage, cover pups with some bedding and reintroduce 
mum “D” in its home cage. 

  11. Put pups “a” in the mum’s “E” cage, cover pups with some bedding and reintroduce mum “E” in its home 
cage.

The following days (PND 2,3,4) the procedure always starts from the mum “A”.
The control manipulation consists in first removing the mother “F” from the cage, than picking up pups “f” and reintroducing them in their home-cage and covering with home-cage bedding. At the end of this manipulation, which lasts 30 sec, the biological mother “F” is reintroduced in the home cage. This manipulation is repeated for the PND1, PND2, PND3 and PND4.
Home cage bedding was changed on postnatal day 10.

Representative data

A general overview of behavioral and physiological outcomes of the RCF protocol in NMRI mice is reported in Luchetti et al. (2015).


Figure 2. Schematic representation of RCF procedure. Pups "a" born from mum "A" spend the first day (D0) with their mum. Then they change caregiver for four consecutive days spending D1 with mum "B", day 2 with dam "C", day D3 with mum "D" and finally from D4 to weaning with adoptive mum "E".

Table 1. Rotation scheme of pups during the repeated cross fostering manipulation. (PND = post-natal day)

MOTHERS
PUPS
PND 0 (birth)
PND 1
PND 2
PND 3
PND 4
A
a
b
c
d
e
B
b
c
d
e
a
C
c
d
e
a
b
D
d
e
a
b
c
E
e
a
b
c
d

Troubleshooting

In some cases (rarely) one/two pups could die/disappear during the days of manipulation and in these cases we usually continue the RCF protocol. To promote adoption, we introduced pups to be adopted in the already present nest of the adoptive dam, and scattered a few amount of her home-cage bedding (and nest material) over them. Following this procedure we never observed pups’ rejection.
We suggest not using the same females for more than one experiment.

Acknowledgments

This study was partly supported by an award granted by the Anna Villa and Felice Rusconi Foundation, by Ministerodell’Università e della Ricerca (PRIN 2008-2010), by Ministerodella Salute (RF-2010-2312059) and by Regione Lazio FILAS for ‘‘Sviluppo della Ricerca sul Cervello’’.

References

  1. Bartolomucci, A., Gioiosa, L., Chirieleison, A., Ceresini, G., Parmigiani, S. and Palanza, P. (2004). Cross fostering in mice: behavioral and physiological carry-over effects in adulthood. Genes Brain Behav 3(2): 115-122.
  2. Battaglia, M., Pesenti-Gritti, P., Medland, S. E., Ogliari, A., Tambs, K. and Spatola, C. A. (2009). A genetically informed study of the association between childhood separation anxiety, sensitivity to CO(2), panic disorder, and the effect of childhood parental loss. Arch Gen Psychiatry 66(1): 64-71.
  3. D'Amato, F. R., Cabib, S., Ventura, R. and Orsini, C. (1998). Long-term effects of postnatal manipulation on emotionality are prevented by maternal anxiolytic treatment in mice. Dev Psychobiol 32(3): 225-234.
  4. Forman, E. M. and Davies, P. T. (2003). Family instability and young adolescent maladjustment: the mediating effects of parenting quality and adolescent appraisals of family security. J Clin Child Adolesc Psychol 32(1): 94-105.
  5. Kendler, K. S., Neale, M. C., Kessler, R. C., Heath, A. C. and Eaves, L. J. (1992). Childhood parental loss and adult psychopathology in women. A twin study perspective. Arch Gen Psychiatry 49(2): 109-116.
  6. Luchetti, A., Oddi, D., Lampis, V., Centofante, E., Felsani, A., Battaglia, M. and D'Amato, F. R. (2015). Early handling and repeated cross-fostering have opposite effect on mouse emotionality. Front Behav Neurosci 9: 93.
  7. Meaney, M. J., Diorio, J., Francis, D., Weaver, S., Yau, J., Chapman, K. and Seckl, J. R. (2000). Postnatal handling increases the expression of cAMP-inducible transcription factors in the rat hippocampus: the effects of thyroid hormones and serotonin. J Neurosci 20(10): 3926-3935.

简介

早期生活事件在编程个体表型中具有关键作用,确实,在婴儿期期间暴露于创伤性经历可以增加各种神经精神病状况(包括情绪和焦虑症)的后来风险。在啮齿动物中的几项研究证明了短期和长期的分离/隔离从照顾者在发育幼崽,对婴儿期和成年期应激的行为和激素反应的影响(D'Amato等人,1998年; Meaney等人,2000; Luchetti等人,2015)。重复交叉繁殖(RCF)是在小鼠幼仔中在产后第四天的生命期间进行的早期操纵。与其他早期操作不同,在RCF治疗的受试者中hypotalamic-pituitary-adrenal(HPA)轴功能不改变。这种操作用于模拟人早期环境不稳定性,这是内化疾病的风险因素,包括分离性焦虑症,恐慌症和CO 2超敏性(Kendler等人,1992; Forman和Davies,2003; Battaglia等人,2009)。

关键字:交叉培养, 早期的环境, 母亲只债券

材料和试剂

注意:见图1.


图1.材料和设备。用于笼养浓缩的纸巾; b。清洁笼C。热板; d。 平衡; e。乳胶手套; F。妈妈和小狗标识的标签。

  1. 纸巾富集纸巾
  2. 手套操作动物(乳胶)
  3. 余额
  4. 10个雌性(最小数目)和5个雄性的相同小鼠品系(我们使用NMRI,C57BL6和DBA,但是在其他菌株中可以使用该方案)用于交配

设备

注意:见图1。

  1. 在"操作步骤"(洗涤的透明高温聚砜笼26.7×20.7×14.0cm,具有干净的床上用品)的点2处,清洁笼子以分离怀孕女性并在操作期间临时保持热板上的幼仔

程序

  1. 交配过程包括住房2女性与一个男性15 d。实验程序要求至少5名女性在24小时内同时分娩。为了达到这个目标并同步雌性发情周期,雌性在交配前几天可能暴露于雄性气味。检查交配时的女性体重。
  2. 在第15天,取出雄性并称重雌性以评估怀孕状态。获得的体重,建议怀孕,取决于所使用的小鼠的株,但通常在15天中为10至15克(交配日体重增加50%);此外,可见的侧向"凸起"在怀孕女性中可见。
  3. 每个怀孕的女性被隔离在与床上用品(纸)的清洁笼子和每日检查幼仔的出生(PND0:出生后第0天)。
  4. 至少5名女性应在24小时内分娩,以允许执行RCF操作。

Rcf操作
小狗与生物母亲一起度过第一个出生日(PND0)。不同于"经典"交叉培养程序(Bartolomucci等人,2004),RCF幼仔在PND1-PND4时间间隔期间每24小时改变护理者4次,通过跟随旋转计划:每个水坝在这个操作期间接收4个不同的窝,每个窝都改变4个不同的收养水坝(收养水坝是妈妈从另一个母亲接受幼仔)(也参见图2和表1)。每日程序包括首先从笼子中取出母亲,然后取出其整个垫料,并立即将该垫料放入不同水坝的家笼中,其幼崽刚刚被移除。然后用收养母亲的家养笼子对RCF幼仔进行半覆盖,然后将其重新引入笼中,并在下一个24小时内留下该幼仔。整个过程持续约30秒,并且在标准光/暗循环(通常在10.30和11.00a.m。之间)的光循环期间的每一天同时发生。每天重复,每次4次,直到达到第四个收养的母亲,其中幼仔离开直到断奶(PND 28)。
收养水坝应该是哺乳期的女性,幼年龄与养育的幼仔相同,幼仔应该有相同或至少相似数量的幼崽,以避免母亲和幼崽可能的营养压力。
控制操作包括首先从笼子中取出母亲,然后拾取对照幼崽,将它们重新引入它们的笼子并用家庭笼子覆盖。在这种持续30秒的操作结束时,将生物母亲重新引入家笼中。该控制过程每天从PND1到PND4进行,以评估RCF协议必然需要的处理过程的可能影响。控制雌性不必在同一天正好分娩。

操作步骤
例如,我们考虑5个RCF和一个CONTROL。我们识别具有大写字母A,B,C,D和E的RCF妈妈,以及具有相同但小写字母a,b的相应幼崽。 c,d和e。控制妈妈识别为F和各个幼犬f。
为了便于该过程,我们在家笼标签上记录妈妈的代码,而小狗的代码被写在较小的可移动标签上,该标签可以被转移(与小狗一起)到收养母亲的笼子标签。
第一天(PND1)

  1. 从主笼中取出妈妈"A"。
  2. 从家笼中取出小狗"a",将它们放在热板(30-35°C)上的清洁笼子中,以保持体温。
  3. 从妈妈的笼子里拿出妈妈"B"。
  4. 将幼仔"b"从他们的笼子里移动到妈妈的"A"笼子里, 半封闭幼崽与床上用品,并重新引入妈妈"A"在其家里的笼子里。
  5. 从其笼子里取出妈妈"C"。
  6. 将幼仔"c"从他们的笼子里移动到妈妈的"B"笼子里,盖上 小狗和一些床上用品,并在其笼子里重新引入妈妈"B"
  7. 从妈妈的笼子里取出妈妈"D"。
  8. 将幼犬"d"从他们的笼子里移动到妈妈的"C"笼子里,盖上 幼仔与一些床上用品,并重新引入妈妈"C"在其家里的笼子里。
  9. 从妈妈的笼子里取出妈妈"E"。
  10. 将幼仔"e"从他们的笼子里移动到妈妈的"D"笼子里,盖上 幼仔与一些床上用品,并重新引入妈妈"D"在其家里的笼子里。
  11. 把小狗"a"放在妈妈的"E"笼子里,用一些床上用品盖上幼崽,并在它的笼子里重新插入妈妈"E"。

接下来的几天(PND 2,3,4),程序总是从妈妈"A"开始。
控制操作包括首先从笼子中取出母亲"F",然后拾起幼犬"f"并将它们重新引入它们的笼子并用家庭笼子覆盖。在持续30秒的该操作结束时,将生物母亲"F"重新引入家笼中。对PND1,PND2,PND3和PND4重复该操作。
家庭笼子床上用品在出生后第10天更换。

代表数据

Luchetti等人(2015)报道了NMRI小鼠中RCF方案的行为和生理结果的一般概述。


图2. R CF procedure 的示意图小狗"从妈妈出生"A"花费第一天他们的妈妈。然后,他们改变护理人员连续四天花费D1与妈妈"B",第2天与坝"C",第D3天,妈妈"D",最后从D4到断奶用收养妈妈"E"。

表1. 在重复交叉繁殖操作过程中幼仔的旋转方案。 (PND =产后天)

母亲
PUPS
PND 0(出生)
PND 1
PND 2
PND 3
PND 4
A
a
b
c
d
e
B
b
c
d
e
a
C
c
d
e
a
b
D
d
e
a
b
c
E
e
a
b
c
d

故障排除

在一些情况下(很少),一两只幼崽在操纵的日子可能死亡/消失,在这些情况下,我们通常继续RCF方案。为了促进收养,我们介绍了在收养水坝已经存在的巢穴中采用的幼仔,并将一些数量的家庭笼子(和巢物)分散在它们上面。按照这个程序,我们从来没有观察到幼仔的排斥。
我们建议不要使用同一个女性进行多个实验。

致谢

这项研究得到了Anna Villa和Felice Rusconi基金会,Ministerodell'Universitàe della Ricerca(PRIN 2008-2010),Ministerodella Salute(RF-2010-2312059)和Regiona Lazio FILAS授予的奖项的部分支持, Sviluppo della Ricerca sul Cervello"。

参考文献

  1. Bartolomucci,A.,Gioiosa,L.,Chirieleison,A.,Ceresini,G.,Parmigiani,S.and Palanza,P.(2004)。 在小鼠中交叉繁殖:成年后的行为和生理性遗留影响。 Genes Brain Behav 3(2):115-122。
  2. Battaglia,M.,Pesenti-Gritti,P.,Medland,S.E.,Ogliari,A.,Tambs,K.and Spatola,C.A。(2009)。 关于儿童分离焦虑,对CO的敏感性(2),惊恐障碍,以及儿童父母亲损失的影响。 Arch Gen Psychiatry 66(1):64-71。
  3. D'Amato,F.R.,Cabib,S.,Ventura,R。和Orsini,C。(1998)。 通过产妇抗焦虑治疗在小鼠中预防出生后操作对情绪的长期影响。 Dev Psychobiol 32(3):225-234。
  4. Forman,E.M。和Davies,P.T。(2003)。 家庭不稳定和青少年青少年失调:育儿质量和家庭安全青少年评估的中介效应。/a> J Clin Child Adolesc Psychol 32(1):94-105。
  5. Kendler,K.S.,Neale,M.C.,Kessler,R.C.,Heath,A.C。和Eaves,L.J。(1992)。 童年家长损失和成年妇女心理病理学。孪生研究视角。 Arch Gen Psychiatry 49(2):109-116。
  6. Luchetti,A.,Oddi,D.,Lampis,V.,Centofante,E.,Felsani,A.,Battaglia,M.and D'Amato,F.R.(2015)。 提前处理和反复交叉对小鼠的情绪有相反的影响。 Front Behav Neurosci 9:93.
  7. Meaney,M.J.,Diorio,J.,Francis,D.,Weaver,S.,Yau,J.,Chapman,K。和Seckl,J.R。(2000)。 产后处理增加大鼠海马中cAMP诱导型转录因子的表达:甲状腺激素的作用和血清素。 J Neurosci 20(10):3926-3935。
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引用:Luchetti, A., Battaglia, M. and D’Amato, F. R. (2016). Repeated Cross-fostering Protocol as a Mouse Model of Early Environmental Instability. Bio-protocol 6(4): e1734. DOI: 10.21769/BioProtoc.1734.
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